Hookworm disease continues to rank among the most important infectious diseases worldwide. Nearly 800 million people are infected with one or both species of hookworms. Blood loss in heavy hookworm infections causes iron-deficiency anemia that is especially devastating in growing children, pregnant women, and the elderly. During infection, the infective larva (L3) encounters a host-specific signal that re-activates its development. This activation of the free-living L3 to the parasitic L3 during infection is a critically important, but poorly understood, event. In the related C. elegans dauer, insulin-like signaling (ILS) and phosphorylation of the transcription factor DAF-16 mediates recovery from developmental arrest. ILS is required for hookworm activation, and a DAF-16 molecule has been identified in hookworms. This project will investigate the basic molecular mechanisms of hookworm L3 activation to parasitism, and specifically the role of insulin signaling and DAF-16, during infection. Aim 1 will investigate the mechanism of DAF-16 regulation during activation of hookworm L3. The phosphorylation status and cellular localization of DAF-16 in non-activated and activated L3 will be determined by cell fractionation and Western blots, and the effect of ILS inhibitors on these parameters established. Aim 2 will determine if ILS signaling through DAF-16 effects expression of activation-associated hookworm genes. Quantitative PCR will be used to measure mRNA levels of known developmentally regulated genes in non-activated, activated and ILS inhibited L3 to determine if they are regulated by DAF-16 and ILS. Aim 3 will determine if insulin signaling and DAF-16 phosphorylation are required for infection. Inhibited L3 will be tested for their ability to infect a permissive host. Insulin signaling and DAF-16 phosphorylation are predicted to be required for successful infection. Relevance: Hookworm control is limited to repeated treatment with anthelmintics drugs, which as a long-term strategy suffers from rapid re-infection and the potential for drug resistance. An effective recombinant vaccine is at least a decade away. The development of new control options, including better antigens and new drug targets, depends on a more detailed understanding of the basic biology of hookworm infection. This project will also provide insight into the basic molecular mechanisms operating during infection and the establishment of parasitic relationships that are relevant to other parasitic nematode infections of human significance.